Change Mechanism Of Hippocampal Angiogenesis In Chronic Stress Elderly Depressive Rats And Intervention Effect Of Venlafaxine

[Objective]To establish elderly depressive rats model by using chronic unpredictable mild stress. And assess the hippocampal angiogenesis changes, related molecular mechanism of elderly depressive rats and intervention effect of antidepressants venlafaxine were evaluated.[Methods]1. According to the table of random number,72SPF healthy elderly (20M) male SD rats were randomly divided into4groups:venlafaxine group, model group, saline group and the control group, with12rats in each group. Rats in Venlafaxine group, model group and saline group were given the chronic unpredictable mild heterogeneous stress with a total of5w. Rats in venlafaxine group were intraperitoneal injected venlafaxine (5mg.Kg-1.d-1) treatment for14d at the end of the3w after stress. Rats in physiological saline group were intraperitoneal injected physiological saline for14d at the same time. Any treatment was nonperformed in control group. The open field test and sucrose consumption test were used to confirm the achievement of the rats model of depression. Meanwhile, the clinical features and the curative effect of venlafaxine in elderly depressive rats model were assessed.2. The changes of hippocampal morphology and hippocampal microvascular density (MVD) of elderly depressive rats in different groups were observed with HE staining and immunohistochemistry respectively.3. To assess the hippocampal expression level of vascular endothelial growth factor (VEGF), angiogenin1(ANG1), basic fibroblast growth factor (bFGF) by using immunohistochemistry. To measure VEGF and receptor VEGFR2, ANG1and receptor TIE2, bFGF and receptor FGFR1protein and gene expression level Using Western Blot (Western Blot) and reverse transcription polymerase chain reaction (RT-PCR) technology. 4. To observe protein and gene expression level of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), tyrosine kinase1(JAK1), cell transcription and transduction activated molecule3(STAT3) using Western blot and RT-PCR. Protein expression level of phospho-Akt (p-Akt) by using Western Blot.5. To evaluate the relationship between MVD and depressive behavior including level scores, vertical scores, cleaning time in open field test and sucrose consumption in sucrose consumption test by using backward method of multiple linear regression analysis. The relationship between VEGF protein and ANG1, bFGF protein was used by bivariate correlation analysis. Bivariate correlation analysis and monadic linear regression analysis were used to evaluate the relationship between MVD and VEGF protein, MVD and ANG1protein, MVD and bFGF protein.[Results]1. The elderly rats depressive model was established successfully in model group, in venlafaxine group and in saline group assessed by open field test and sucrose consumption test. Open field test:Intra-group comparison:Compared with that of the same group1day before stress, results showed that level scores, vertical scores were lower in model group, in venlafaxine group and in saline group at the end of3w,5w after stress(P<0.05), and cleaning time was shortened in these three groups (P<0.05); Compared with that of the same group at the end of3w after stress, results showed that level scores, vertical scores were lower in model group and in saline group at the end of5w after stress(P<0.05), and cleaning time was shortened in these two groups(P<0.05). Comparison among groups:Compared with control group, level scores and vertical scores were lower(P<0.05), and cleaning time was shortened in venlafaxine group at the end of5w after Stress (P<0.05); Level scores and vertical scores were markly lower(P<0.01), and cleaning time was significantly shortened in model group and in saline group (P<0.01). Vertical scores in venlafaxine group were higher than that in saline group (P<0.05). Sucrose consumption test:Intra-group comparison:Sugar consumption, sucrose preference in model group and in saline group at the end of5w after stress were less than that of the same group1day before stress and at the end of3w after stress(P<0.05). Comparison among groups:At the end of5w after Stress, compared with control group, sucrose consumption in model group and in saline group were significantly reduced (P<0.01). Compared with saline group, sucrose consumption in venlafaxine group was increased (P<0.05).2. HE staining showed that hippocampal pyramidal cells were well-organized structures in vitro, with a thick layer in control group, accompanied by normal blood capillary quantity. Hippocampal pyramidal cells layer was thiner in model group and saline group, with cells gap increasing, and loose structure in disorder, blood capillary quantity reducing. Hippocampal pyramidal cells layer in venlafaxine group was thin, but cells were close together in alignment, a significant increase in blood capillary numbers was found, with irregular lumen. Compared with control group, hippocampal microvessels density in saline group and in model group were decreased (P<0.05). Compared with saline group, hippocampal microvascular density was increased in venlafaxine group (P<0.05).3. Compared with control group, expression of hippocampal VEGF and VEGFR2protein, VEGFmRNA and VEGFR2mRNA was decreased in model group and in saline group respectively (P<0.05). Compared with saline group, the expression of hippocampal VEGF and VEGFR2protein, VEGFmRNA and VEGFR2mRNA was increased in venlafaxine group respectively (P<0.05).4. Compared with control group, expression of hippocampal ANG1and TIE2protein, ANG1mRNA and TIE2mRNA in saline group and in model group was increased (P<0.05). Compared with saline group, expression of hippocampal ANG1and TIE2protein, ANG1mRNA and TIE2mRNA in venlafaxine group was increased (P<0.05).5. Compared with control group, expression of hippocampal bFGF and FGFR1protein, bFGFmRNA and FGFRlmRNA was decreased in model group and in saline group (P<0.05). Compared with saline group, expression of hippocampal bFGF and FGFR1protein, bFGFmRNA and FGFRlmRNA was significantly increased in venlafaxine group (P<0.01).6. Compared with control group, hippocampal PI3K and Akt protein, p-Akt protein, PI3KmRNA and AktmRNA expression in saline group and in model group were decreased (P<0.05). Compared with saline group, hippocampal PI3K and Akt protein, p-Akt protein, PBKmRNA and AktmRNA expression were significantly increased in venlafaxine group (P<0.01).7. There was no statistical significance in hippocampal JAK1and STAT3proteins, JAK1mRNA and STAT3mRNA expression in four groups (P>0.05).8. The coefficient of the vertical scores in open field test had statistical significance between MVD and depressive behavior in multiple regression analysis (P<0.05). The regression equation was MVD=5.056+0.580vertical score. In bivariate correlation analysis between VEGF protein, ANG1protein and bFGF protein, VEGF protein was related to ANG1protein, VEGF protein was associated with bFGF protein(r=0.573,0.742, P=0.00,0.000). In monadic linear regression analysis between MVD and VEGF protein, the coefficient of VEGF protein had obvious statistical significance (P<0.01), the regression equation was MVD=3.391+11.507VEGF. In monadic linear regression analysis between MVD and bFGF protein, the coefficient of bFGF protein had distinct statistical significance (P<0.01), the regression equation was MVD=3.047+10.884bFGF.[Conclusions]1. A successful elderly depressive rat model was established. Mental retardation symptom was outstanding, and this symptom aggravated over time. Falling interest symptom appeared to come later. The curative effect of the same dose of venlafaxine was different according to different symptoms of elderly rats. Venlafaxine could relieve the mental retardation symptom aggravating, but this symptom could not return to normal level treated by venlafaxine. Falling interest symptom was prevented and controlled by using venlafaxine.2. Hippocampal microvessels density in elderly depressive rats were decreased, angiogenesis could be inhibited. The hippocampal microvessels density was increased and hippocampal angiogenesis could be promoted after venlafaxine intervention. Hippocampal microvascular density in geriatric depressive rats was associated with the depressive symptoms of senile rats. It was suggested that elderly depressive disorder could be associated with vascular injury. 3. Expression level of hippocampal angiogenines and their receptors protein and gene changed in elderly depressive rats after chronic stress. Venlafaxine could promote the angiogenines and receptors protein and gene expression. Hippocampal VEGF protein was related to ANG1protein, VEGF protein was associated with bFGF protein. Hippocampal microvascular density was related to expression of VEGF protein, hippocampal microvascular density was associated with expression of bFGF protein as well. It was suggested that VEGF, ANG1and bFGF were involved in the pathogenesis of elderly depressive disorder and antidepressant effect of venlafaxine in the mechanism of angiogenesis.4. Hippocampal PI3K/Akt signaling pathway could be affected in elderly rats depression model after chronic stress, venlafaxine could promote PI3K/Akt signaling pathways activation. JAK1/STAT3signaling pathway had not altered obviously. It was suggested that PI3K/Akt signaling pathway in the mechanism of angiogenesis was involved in the pathogenesis of elderly depressive disorder and the antidepressant effect of venlafaxine.